It is often desirable to detect or quantify very small concentrations of nucleic acids in biological samples. Typically, to perform such measurements, the nucleic acid in the sample (i.e., the target nucleic acid) is hybridized to a detection oligonucleotide that contains at least ten contiguous nucleotides that are homologous to a portion of the target nucleic acid in the sample. In order to obtain a detectable signal proportional to the concentration of the target nucleic acid, either the target nucleic acid in the sample or the detection oligonucleotide is associated with a signal generating reporter element, such as a radioactive atom, a chromogenic or fluorogenic molecule, or an enzyme (such as alkaline phosphatase) that catalyzes a reaction that produces a detectable product. Numerous methods are available for detecting and quantifying the signal.
Following hybridization of a detection oligonucleotide with a target, the resulting signal-generating hybrid molecules must be separated from unreacted target and detection oligonucleotides. In order to do so, many of the commonly used methods immobilize the target nucleic acids or detection oligonucleotides on solid supports. This permits fractionation and identification of the hybridized nucleic acids. The target nucleic acid may be captured by oligonucleotides immobilized on solid supports. More frequently, so-called "sandwich" hybridization systems are used. These systems employ capture oligonucleotide covalently attached to a solid support for capturing detection oligonucleotide-target nucleic acid adducts formed in solution. Solid supports with linked oligonucleotides may also be used in methods of affinity purification.
Presently available solid supports to which oligonucleotides are linked include nitrocellulose or nylon membranes, activated agarose supports, or diazotized cellulose supports. Oligonucleotides containing an aldehyde or carboxylic acid group at the 5'-terminus have been covalently attached to non-porous polystyrene latex solid microspheres (Kremsky et al. (1987) Nucl. Acids Res. 15:2891). Although this method provides good end-attachment results, it is disadvantageous in that, at the end of the coupling reaction, non-covalently bound oligonucleotide must be removed by a tedious gel electrophoresis step. The presently available supports with attached oligonucleotides, however, have several other disadvantages in practice. Either the bonds between these supports and the oligonucleotides are not covalent, which allows release of the oligonucleotides from the support, or the supports have other shortcomings. For example, N-hydroxysuccinimide or cyanogen bromide activated polysaccharide affinity supports have a serious drawback in the leakage of ligands. This not only leads to misleading results but, even more importantly, poses health hazards when immunoaffinity-purified products produced by recombinant DNA synthesis are complexed with mouse monoclonal antibodies (see e.g., Wilchek et al. (1987) Biochemistry 26:2155 and Wilchek et al. (1975) Proc. Natl. Acad. Sci. USA 72:1055). Leakage of ligands from solid supports can also interfere with affinity purification. If the free ligand that leaks from the support is more effective as a binder than the insolubilized ligand, the free ligand will bind the target macromolecule essentially irreversibly, and prevent affinity adsorption to the support. Further, cyanogen bromide activation of polysaccharide supports leads to the formation of N-substituted isoureas on the surface of the matrix. These confer undesirable ion exchange properties on the support, which become problematic in affinity chromatography, when analytes, such as nucleic acids, are present in very minute concentrations.
Solid supports with linked oligonucleotides are also used in bead-based sandwich hybridization system (BBSHS) (see, e.g., EP 276,302 and Gingeras et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173). According to this method, target nucleic acid and an oligonucleotide probe used for its detection, which is complementary to at least a region of the target, are hybridized. The obtained adduct is then captured by a second oligonucleotide that is complementary to a different region of the target and is end-attached to a solid support. The amount of the detection oligonucleotide associated with the solid support is directly related to the amount of the target captured. In this way, the BBSHS can be used to determine the amount of a single-stranded nucleic acid in a sample. Such assays typically use radioactively (e.g., .sup.32 P) labeled cloned DNA or synthetic oligonucleotides.
The potential sensitivity of assays that use support-bound oligonucleotides is a function of numerous parameters, including the level of non-specific background. In the presence of target, the non-specific background can result from various factors, including (1) hybridization of the detection and capture oligonucleotide to non-complementary sequences of the target nucleic acid; (2) hybridization of the detection oligonucleotide to the capture oligonucleotide; and (3) non-specific attachment of the detection oligonucleotide to the bead support or walls of the reaction vessel. The first two factors can be minimized by sufficiently stringent solution hybridization, capture, and wash conditions. The third factor (i.e., non-specific binding, including the percentage of oligonucleotides that are end-attached) appears to be an inherent property of the support and detections system used. In case of the conventionally used solid supports, particularly Sephacryl.TM. dextran beads, the percentage of oligonucleotides that are end-attached to the support is relatively low, about 50-55% (see, e.g., Ghosh et al. (1987) Nucl. Acids Res. 15:5353). A higher degree of end-attachment, however, is highly desirable because it would be result in greater capture potential of the immobilized oligonucleotide probe that would improve the sensitivity of assays in which such supports are used. Methods for improving the percentage of end-attachment and decreasing the level of non-specific attachment of the detection oligonucleotide to the bead support or walls of the reaction vessel are not presently available.
Therefore, it is an object herein to provide a method for preparing solid supports in which a substantial portion of the attached oligonucleotides are attached via their 5'-ends. It is also an object herein to provide solid supports in which a substantial portion of the attached oligonucleotides are attached via their 5'-ends.